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TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer

ABSTRACT: Human telomerase reverse transcriptase (hTERT) is a rate-limiting catalytic subunit of the enzyme telomerase, which plays a central role in maintaining replicative immortality in normal stem cells and many cancer cells. Mostly silenced in normal somatic cells, hTERT is reactivated in cancer commonly through highly recurrent hot spot point mutations in the hTERT promoter. Bladder cancer has a 60-70% hTERT promoter mutation prevalence, commonly at -124 bp from the translation start site, and this is associated with increased hTERT expression and poor patient prognosis. Identifying targetable signaling pathways that drive hTERT transcription from the mutant promoter could lead to therapies with reduced effects on normal progenitors. Here we describe a new platform that probes endogenous hTERT regulation by allele-specific insertion of GFP at either wild-type or mutant promoter allele within human bladder cancer cells harboring a heterozygous mutation. Using cells expressing the GFP-hTERT fusion protein, we performed a pooled CRISPR-Cas9 Kinome KO genetic screen to identify regulators of mutant hTERT promoter activity. We identified TRIM28 as an activator and TRIM24 as a suppressor of hTERT expression. Similar to hTERT, TRIM28 expression stratifies patient outcome and inhibits promotes cell growth both in vitro and in vivo, supporting their clinical relevance. TRIM28 and TRIM24 get recruited to the mutated promoter through GABPA, where TRIM24 interacts with TRIM28 and inhibits its activity as a transcriptional activator. TRIM28 phosphorylation causes its release from TRIM24 and activates hTERT transcription. mTOR phosphorylates TRIM28 through the mTORC1 complex leading to hTERT expression, while mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses the phosphorylation and promoter binding of TRIM28, resulting in reduced hTERT expression and lower cell viability. This study uses a bespoke platform to identify a novel therapeutically tractable regulatory pathway for cancers harboring mutant hTERT promoters.

ORGANISM(S): Homo sapiens

PROVIDER: GSE181461 | GEO | 2021/08/06

REPOSITORIES: GEO

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Project description:Recent genetic studies in mice have established a key role for the nuclear receptor coregulator Trim24 in liver tumor suppression and provided evidence that Trim24 suppresses hepatocarcinogenesis by inhibiting retinoic acid receptor alpha (Rara)-dependent transcription and cell proliferation. However, it is unknown which downstream targets of Rara regulated by Trim24 are critical for tumorigenesis. We report here that loss of Trim24 results in the overexpression of interferon (IFN)/STAT pathway genes in the liver, a process that occurs early in tumorigenesis and is more pronounced in tumors, despite the enhanced expression, late in the disease, of negative regulators such as Usp18, Socs1 and Socs2. Remarkably, Rara haplodeficiency, which was previously shown to suppress tumor development in Trim24-/- mice, also suppresses overexpression of the IFN/STAT pathway, thus providing evidence for a cross-pathway control that may be relevant to the transformation process. Biochemical studies revealed that Trim24 binds to the retinoic acid (RA)-responsive element in the Stat1 promoter in a RA-dependent manner and represses RA-induced transcription from this promoter. Together, these results identify Trim24 as a novel regulator of the IFN/STAT pathway and indicate that Trim24-mediated repression of the IFN/STAT signaling through Rara inhibition may play a critical role in preventing liver cancer. Generation of Trim24-/- mice has been described previously (Khetchoumian et al., 2007) by gene disruption. To generate compound mutant mice with a single allele of Rara deleted in the Trim24 -/- mutant background, we crossed Trim24 -/- mice with Rara+/- mice. The resulting Trim24+/- Rara+/- mice were generated in the hybrid (C57BL/6 (60%), 129/Sv (40%)) genetic background. These double heterozygous Trim24+/- Rara+/- mice were intercrossed to generate Trim24 -/-, Trim24 -/- Rara+/- and wild-type mice. Transcriptional profiling of mice at 5-weeks and 14-weeks of age.

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TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer

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